neurosteroids, Gabaa receptors and neurosteroid

Tvrdeić et al.

Review

Neurosteroids, GABAA receptors and neurosteroid based drugs: are we witnessing the dawn of the new psychiatric drugs? Ante Tvrdeić1, Ljiljana Poljak2 1 Department of Pharmacology, University of Zagreb School of Medicine, Šalata11, Zagreb, Croatia 2 Department of Physiology and Immunology, University of Zagreb School of Medicine, Šalata, Zagreb, Croatia

Corresponding author: Associate Professor Ante Tvrdeić, Ph.D., Department of Pharmacology, University of Zagreb School of Medicine, Šalata11, Zagreb, Croatia, E-mail address: ante.tvrdeic@ mef.hr Doi: 10.21040/eom/2016.2.7 Received: January 14th 2016 Accepted: February 20th 2016 Published: March 14th 2016

Copyright: © Copyright by Association for Endocrine Oncology and Metabolism. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc-nd/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Funding: this work was supported by research grants from the Ministry of Science, Education and Sports, Republic of Croatia. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

60 Endocrine Oncology and Metabolism

Conflict of interest statement: The authors declare that they have no conflict of interest. Data Availability Statement: All relevant data are within the paper. Abstract In broad biological terms, neurosteroids can be defined as a class of endogenous steroids synthesized in the brain or in peripheral steroidogenic tissues having potent and relatively selective activity on brain gamma-aminobutyric acid A (GABAA) receptors. In this regard, the most important neurosteroids are allopregnanolone and allotetrahydrodeoxycorticosterone (allo THDOC). These α-reduced derivatives of pregnenolone and progesterone act as positive allosteric modulators of GABAA receptors. As such, they potentiate the inhibitory action of GABA on GABAA receptors and produce a wide spectrum of behavioral actions ranging from anxiolytic, anticonvulsive, sedative, hypnotic, amnestic (loss of memory), myorelaxant, and anesthetic effects. Sulfated derivatives of pregnenolone and dehydroepiandrosterone ,pregnenolone sulfate (PS) and dehydroepianddrosterone sulfate (DHEAS), are also very important neurosteroids. In contrast to allopregnolone and alloTHDOC, PS and DHEAS induce excitatory effect on neurons because they facilitate the block of GABAA receptors. The spectrum of behavioral effects of PS and DHEAS consists of analeptic, anxiogenic, proconvulsive, and anamnestic (cognitive enhancing). The purpose of this review paper is to analyze recent research in the field of neurosteroids and neurosteroid-based drugs with emphasis on interaction of neurosteroids with brain GABAA receptors. This article also provides an overview of neurosteroids-based strategies for the development of innovative therapeutic approaches. GABAA receptor modulating steroids (GAMS), GABAA receptor modulating steroid antagonists (GAMSA), and translocator protein (TSPO) activators are examples of innovative therapeutic approaches in treating clinically important neurological and psychiatric diseases. Consequently, the therapeutic potential of GAMS, GAMSA, and TSPO activators will be briefly evaluated. Key words: Neurosteroids; GABAA receptors; translocator protein; drugs

Tvrdeić et al.

1. Introduction By definition, neurosteroids are steroid molecules synthesized within the brain under physiologic conditions, and released in a paracrine or autocrine way to induce fast (nongenomic) or long-lasting (genomic) modulation of nerve cell function [1]. The term neuroactive steroid is usually reserved for steroids that act on the central or peripheral nervous system, wherever they come from: by exogenous application (drugs), from steroidogenic tissues, by endocrine secretion (hormones), or from local nerve tissues (neurotransmitters and neuromodulators). For simplicity, in this review, the term neurosteroids and neuroactive steroids will be used as interchangeable terms. In this review, we will focus on neurosteroids, neurosteroid-based drugs, and the interaction of neurosteroids with GABAA receptors to produce rapid, nongenomic modulatory effects on brain cells. Genomic effects of (neuro)steroids mediated by classical hormonal receptors, as well as nongenomic, rapid effects, other than the one mediated by GABAA receptors are reviewed in details elsewhere.

2. Biosynthesis of neurosteroids and sites of drug action The biosynthesis of neurosteroids in the brain tissue is very similar to the synthesis in any other steroidogenic organ - ovary, testis, placenta, or adrenal gland. Neurosteroidogenesis is detected in both, neurons [2] as well as in glial cells [3]. The first phase of neurosteroid synthesis takes place inside the mitochondria. The mitochondrial phase of steroidogenesis yields pregnenolone as a final metabolic product. Pregnenolone arises from cholesterol (Figure 1.) by the action of cytochrome P450 enzyme 11A1 (CYP11A1), also known as CYPscc (2). Lipophilic cholesterol is not able to freely pass through the aqueous environment in order to reach the inner mitochondrial membranes. Thus, cholesterol must be translocated through the hydrophilic media by a process that involves a specific transport system. By the coordinated action of translocator protein (TSPO; for details see text bellow) and steroidogenic acute regulatory protein (STARD1), cholesterol is transported from the outer to the inner mitochondrial membrane [4, 5]. Enzyme CYP11A1, which is responsible for pregnenolone synthesis, is located close to the inner mitochondrial membrane. This enzyme removes the side chain from the

27-carbon ring of cholesterol and produces the 21-carbon ring steroid molecule of pregnenolone. Although pregnenolone produces behavioral effects on its own [5], it is much more important as a precursor of the all other endogenous neurosteroids. Since the transport of cholesterol to the inner mitochondrial membrane is “bottleneck” in the production of pregnenolone, and pregneneolone is the “mother” of all neurosteroids, translocation of cholesterol from the outer to inner mitochondrial membrane is considered to be a rate-limiting step in the synthesis of neurosteroids [6]. The neurosteroid precursor pregnenolone, is further metabolized in the endoplasmic reticulum by multiple, subsequent enzymatic reactions (microsomal phase of neurosteroid synthesis). As it is shown in Figure 1., these reactions are catalyzed with a variety of enzymes (CYP450 as well as non CYP450 types) and are able to produce functionally and structurally diverse neurosteroids [7-9]. For example, pregnenolone (Figure 1., white box) is conjugated by the enzyme sulfotransferase (SULT2A1) yielding pregnenolone sulfate (Figure 1., PREGN.S, red box), the neurosteroid with excitatory action on nerve cells [9, 10]. The biochemical reactions and pathways involved in neurosteroid synthesis are usually bidirectional. In the case of excitatory pregnenolone sulfate, a reverse reaction catalyzed by sulfatase, returns inhibitory acting pregnenelone [6]. Also, in two subsequent reactions catalyzed by CYP17A1, pregnenolone yields dehydroepiandrosterone (Figure 1., DHEA, red box), another active neurosteroid that seems to have a paradoxical, dual mode of action on neurons [11]. Namely, DHEA induces an excitatory response when it is used acutely. On the contrary, when DHEA is used chronically, inhibitory effects on neurons should be expected. DHEA is sulfated by sulfotransferase to produce dihydroepiandrosterone sulfate (Figure 1., DHEAS, red box), a neurosteroid with pure excitatory action on neurons [12]. By removing sulfate from the DHEAS molecule with the enzyme sulfatase, DHEAS is metabolized back into DHEA. From DHEA, by the action of 3α- hydroxysteroid dehydrogenase (3α-HSD) or 5-α and 5-β reductase [13], the endogenous neurosteroids 5α,3α- androsterone (Figure 1., ANDROSTERONE, blue box) and 5β,3α-androsterone (also known as ethiocolanolone) are produced. These metabolites of DHEA act as inhibitory neurosteroids [14, 15]. Also, the pheromone androstenol, could

Endocrine Oncology and Metabolism

61

Tvrdeić et al.

be synthesized from DHEA in human and boar testicles [16]. This lipophilic and musk smelling substance, acts on GABA-A receptors located in the olfactory bulb to increase sexual receptive behavior [16]. As an interesting coincidence, it must be emphasized that two Nobel Prize laureates from Croatia, Vladimir Prelog and Lavoslav Ružička, were pioneers of androstenol isolation from boar testicles [17]. Progesterone (Figure. 1, white box) is synthesized from pregnenolone in a reduction reaction, which is catalyzed by 3β- hydroxysteroid dehydrogenase or 3β-HSD [7]. From a historical perspective, progesterone had a fundamental role in the development of the neurosteroid concept. Namely, a report published by Hans Selye on the anesthetic effect of progesterone in rats and mice was the first experimental evidence for the rapid central nervous system (CNS) effect of steroid molecules [18]. Like pregnenolone, progesterone also serves as a precursor for the synthesis of important neurosteroids. In two subsequent metabolic steps catalyzed by 5-α reductase and 3α- or 3β-HSD, the following neurosteroids are derived from progesterone: 3α,5α tetrahydroprogesterone (known as allopregnanolone; Figure 1., 3α5αTH-PROGEST., blue box), 3α,5β- tetrahydroprogesterone (known as pregnanolone,), 3β,5α- tetrahydroprogesterone (known as isoallopregnanolone), and 3β,5β-tetrahydroprogesterone (known as epiallopregnanolone). Both 3α tetrahydroprogesterone molecules produce inhibitory actions on neurons. On the other hand, 3β tetrahydroprogesterone molecules are natural excitatory neurosteroids with the ability to antagonize the anesthetic effect of inhibitory neurosteroids, including allopregnanolone [19-21]. Important groups of inhibitory acting neurosteroids produced from progesterone are derivatives of deoxycorticosterone. In a three step reaction catalyzed by CYP21, 5-α reductase, and 3α-HSD, with deoxycorticosterone and dihydrodeoxycorticosterone (Figure 1., 5αDH-DEOXYCORT., blue box) as intermediate products, progesterone yields 3α,5α-tetrahydrodeoxycorticosterone (alloTHDOC; Figure 1., 3α5α TH-DEOXYCORT., blue box) and 3α,5β- tetrahydrodeoxycorticosteroneor THDOC [6, 22]. Both reduced derivatives of deoxycorticosterone produce inhibitory effects on neuronal cells [23, 24]. It is interesting that protein or mRNA for the CYP21 enzyme, which is necessary for the production of deoxycorticosterone from progesterone, and for the production of deoxycortisol from 170H- progesterone, have


Figure 1. A schematic diagram of neurosteroidogenesis

with selected enzymes and enzyme inhibiting drugs. Synthesis of neurosteroids is not complete, for the sake of visibility. Some neurosteroids that are missing here are mentioned in the text (ethiocolanolone and androstenol, DHEA metabolites; pregnanolone and 3β tetrahydroprogesterones, progesterone metabolites; THDOC, deoxycorticosterone metabolite). Neurosteroids with inhibitory actions on neurons are presented in blue boxes. Sulfated neurosteroids with excitatory actions on neurons are presented in red boxes. DHEA has dual mode of action, depending on the way of use. Acute effect of DHEA is excitation of neurons. Thus, DHEA is presented in form of red box. Abbreviations: TSPO = translocation protein; STRD1 = steroidogenic acute regulatory protein; PREGN.S. = pregnenolone sulfate; SULT2A1 = sulfotransferase; 17OH-PREGN. = 17 hydroxypregnenolone; DHEA = dehydroepiandrostetrone; DHEAS = dehydroepiandrosterone sulfate; 3α-HSD or 3β-HSD = hydroxysteroid dehydrogenase; 5α-RED. = 5α-reductase; 5αDH-PROGEST. = 5α dihydroxyprogesterone ; 3α5αTH-PROGEST. = 3α5α tetrahydroprogesterone, also known as alloprogesterone; 17OH-PROGEST. = 17 hydroxyprogesterone; ANDROSTENED. = androstenedione; 5αDH-DEOXYCORT. = 5α dihydro deoxycortycosterone; 3α5α TH-DEOXYCORT. = 3α5α tetrahydro deoxycorticosterone; AROM. = aromatase, also known as CYP19A1 or P450arom; abirat. a. = abiraterone acetate; finast. = finasteride; MPa = medroxyprogesterone acetate

Tvrdeić et al.

not yet been detected in brain tissue [6]. On the other hand, mRNA and proteins for CYP11B and CYP11AS, which are responsible for cortisol and aldosterone synthesis, respectively, were found in a significant amount in the cortex, hippocampus, basal ganglia, cerebellum (CYP11B), and hypothalamus (CYP11AS) [6]. Specific drugs could manipulate the synthesis of both excitatory and inhibitory neurosteroids. For example, the biosynthesis of inhibitory acting neurosteroids can be inhibited by 5α-reductase inhibitors (Figure 1., 5α-RED.) such as finasteride (13,25) or by inhibitors of 3α-HSD such as medroxyprogesterone acetate [26]. Inhibition of inhibitory neurosteroids with finasteride or medroxyprogesterone acetate (Figure 1., MPA) has been associated with depression, anxiety, irritability, and sexual dysfunction. These behavioral alternations are well known side effects of 5α-reductase inhibitors [27, 28] or MPA [29, 30]. Inhibitors of CYP11A (aminoglutethimide and ketoconazole), by blocking the production of pregnenolone (Figure 1), may inhibit the production of all neurosteroids [31, 32]. Inhibitors of CYP17A1, such as abiraterone acetate (Fig. 1; antiandrogen), may block the production of DHEA from pregnenolone [33] and, indirectly, the production of its derivatives DHEAS (excitatory neurosteroid) and androsterone (inhibitory neurosteroid). The CYP11B1 inhibitor metyrapone (anticortisol drug) may inhibit the synthesis of potential excitatory neurosteroids, which are reduced metabolites of cortisol [6, 34]. Trilostan inhibits 3β-HSD (Figure 1) and conversion of pregnenolone to progesterone [35]. Thus, trilostan inhibits the production of all classical steroid hormones – progesterone, estrogens, androgens, glucocorticoids, and mineralocorticoids. Likewise, trilostan inhibits the production of all brain neurosteroids, with the exception of pregnenolone and its derivatives PS, DHEA, DHEAS, and the DHEA derivative androsterone. Synthesis of these neurosteroids does not depend on the 3βHSD enzyme. Aromatase (Figure 1., AROM) is the enzyme that catalyzes the conversion of androgens to estrogens [35]. In mice, letrozole (Fig. 1), an aromatase inhibitor that blocks the conversion of testosterone to 17β-estradiol, significantly decreased testosterone potentiation of pentylentetrazole-induced seizures. Thus, endogenously produced 17β-estradiol derived from testosterone might contribute to the observed pro-convulsive actions of

testosterone in mice [37]. Also, specific drugs could activate steroidogenic enzymes. The term “selective steroidogenic brain stimulants” (SSBSs) has been proposed for drugs that activate steroidogenesis and potentiate the effects of endogenously synthesized neurosteroids [38]. Good examples of SSBSs are fluoxetine and norfluoxetine. Fluoxetine is a well-known antidepressant drug that belongs to a class of selective serotonin reuptake inhibitors (SSRIs), and norfluoxetine is an active metabolite of fluoxetine. Fluoxetine, norfluoxetine, several other SSRI (paroxetine, sertraline), and other non-SSRI antidepressant drugs (venlafaxine, mirtazapine) are potent inducers of the 3α-HSD enzyme [39]. These drugs have been found to increase levels of inhibitory neurosteroid allopregnenalone and to potentiate its behavioral effects [40]. The TSPO activators, a new group of investigational drugs that have the potential to treat important neurological and psychiatric diseases [41] also increase brain levels of inhibitory neurosteroids and potentiate their behavioral action (for details about TSPO activators, see text below).

3. GABAA receptors as a target for the nongenomic action of neurosteroids 3.1. GABAA receptors – how many binding sites are there? The GABAA receptor is a protein complex made up of five protein subunits and associated with the chloride channel (Figure 2). Each protein subunit (Figure 2., left) contains four transmembrane regions, one N-terminal extracellular domain with a characteristic cysteine loop (cys-loop), and one intracellular loop and one extracellular COOH terminus [42]. Protein subunits of GABAA receptors are symmetrically arranged around the chloride channel, building the wall of this channel (Figure 2, right). The main inhibitory neurotransmitter, γ-aminobutyric acid (GABA), by acting on its binding site at the GABAA receptor, opens the chloride channel and increases the transmembrane inflow of chloride anions, inducing a rapid (in milliseconds), inhibitory, hyperpolarization potential on neurons. Due to its typical structure and function, the GABAA receptor belongs to a family of cys-loop ligand gated ion channel (LGIC) receptors [43]. Also, the GABAA receptor is a receptor complex, which


63

Tvrdeić et al.

Figure 2. Structure of the GABAA receptor Left: GABAA

monomeric subunit imbedded in a lipid bilayer (yellow lines connected to blue spheres). The four transmembrane α-helices (1–4) are depicted as cylinders. The disulphide bond in the N-terminal extracellular domain that is characteristic of the family of cys-loop receptors (which includes the GABAA receptor) is depicted as a yellow line. Right: Five subunits symmetrically arranged about the central chloride anion conduction pore. The extracellular loops are not depicted for the sake of clarity.

is composed of multiple binding sites. It is generally accepted that this receptor complex consists of the following binding sites: binding sites for the neurotransmitter GABA and competitive antagonists [44-47], modulatory binding sites for sedative/hypnotic and anxiolytic benzodiazepines [48-51], binding sites for sedative/hypnotic and anesthetic barbiturates [52-54], binding sites for convulsive toxins like picrotoxine and pyrethroid insecticides [55, 56], and neurosteroid modulatory sites [5760] for neurosteroids. The existence of these “classical” binding sites is consistently and repeatedly confirmed by different research groups all over the world. In almost 70 years of research devoted to GABA neurotransmission [62-66], evidence on “classical” GABAA receptor binding sites has been accumulated at different levels of body organization (organism, tissue, cellular, and subcellular level) and by using of various research methods, including behavioral (in vivo testing in models for anxiety, depression, sedation/hypnosis, epilepsy), biochemical (receptor binding studies and 36chlorine uptake in synpatosomes), electrophysiological (“patch clamp” and similar techniques), and molecular biology (receptor subunit transfection, gene knockout, gene knockdown, site directed mutagenesis) methods [64-66]. Despite this, more than a dozen binding sites, other than the “classical” ones mentioned above, were postulated to exist within GABAA receptors. Just to mention few:


binding sites for general anesthetics and ethanol [67-70], binding sites for furosemid and amiloride diuretics [71, 72], binding sites for Zn2+ [73], binding sites for fluoroquinolone antibiotics [74], binding sites for fenamate NSAIDs [74, 75], and binding sites for dehydrogenated ergot drugs [76-78]. The 2015 Nobel Prize in Physiology or Medicine was awarded with one half jointly to W.C. Campbell and S. Ōmura for their discovery of broad spectrum antihelmintic drugs, avermectine, and ivermectin (79). A long time ago, Wang and Pong proposed the mechanism of action of those two drugs which involved the interaction of the anthelmintic drug with the specific binding site at the GABAA receptor complex in invertebrate muscles [80]. Only recently, EstradaMondragon and Lynch published results that confirm the direct interaction between ivermectin and vertebrate GABAA receptors [81].

3.2. GABAA receptor assembly and GABAA receptor subtype heterogeneity So far, eight GABAA receptor subunits (named by the Greek alphabet - alpha, beta, gamma, delta, eta, omega, pi, and rho) and their 19 isoforms (α1-6, β1-3, γ1-3, δ, ε, θ, π, ρ1-3) have been identified [82]. From these, theoretically ≈800 different hetero or homo pentamer subunit combinations and structurally different GABAA receptors are possible to build. However, only 11 subunit combinations were positively identified as native GABAA receptors in mammalian brain. With 9 GABAA receptor subunit combinations in the category “highly probable” and another 6 in the category “tentative”, that makes the final list of 26 GABAA receptors. The most abundant native GABAA receptor combination is the α1β2γ2 (60% of all). GABAA receptors which are assembled of α2β3γ2 and α3βxγ2 subunit combinations represent 15–20% and 10–15% of the total native receptors, respectively [83]. 3.3. Modulation of GABAA receptors by neurosteroids Excitatory neurosteroids (PS, DHEAS, DHEA if acutely applied, isoallopregnanolone, and epipregnanolone) act as negative allosteric modulators of GABAA receptors and facilitate closing of GABAA receptor associated Cl- ionophore [5, 10, 12, 84]. In contrast, inhibitory neurosteroids (allopregnanolone, pregnanolone, THDOC, alloTHDOC, androsterone, DHEA if chronically applied, ethiocholanolone, and androstenol) act as positive allosteric modulators of GABAA receptors and

Tvrdeić et al.

potentiate opening of GABAA receptor associated Clchannels by GABA [14, 15, 19-21, 23, 85, 86]. GABAA receptor binds two GABA molecules, at the interface between the α and β subunit. This means that the cooperative action of two GABA molecules is needed for the GABA gate chloride channel. Binding of benzodiazepines (i.e. diazepam) at the interface of α and γ subunits induces positive allosteric modulation of GABAA receptors and facilitates opening of chloride channels by GABA. In the model of neurosteroid binding proposed by Hosie [87], four molecules of neurosteroids interact with the GABAA receptor. Two neurosteroid molecules interact with GABA binding sites to potentiate the opening of Cl channels with GABA. The other two molecules interact with the hydrophobic M region to directly open Cl- channels. Thus, at low concentrations, neurosteroids potentiate the effect of neurotransmitter GABA at GABAA receptors. At high doses, neurosteroids, like barbiturates, are able to directly open GABAA receptor associated chloride channels. It seems quite possible that neurosteroid binding sites consists of multiple, overlapping binding sites, one potentiating binding site, one activating binding site, and one binding site for excitatory (sulfated) neurosteroids [58, 88]. GABA receptors consisting of α1/β subunits or ρ subunits are relatively insensitive to neurosteroids [89]. In contrast, GABAA receptors containing δ subunit, particularly in combination with α4 or α6 subunits are highly sensitive to low, nanomolar concentrations of neurosteroids [90, 91].

4. Behavioral effects of neurosteroids Inhibitory neuroactive steroids induce dose dependent anxiolytic, anticonvulsive, sedative/hypnotic, amnestic (high dose), and anesthetic effects [92]. The spectrum of activity of inhibitory neuroactive steroids is, therefore, very similar to those of classical benzodiazepines, which are full agonists at benzodiazepine receptors. However, in comparison to full agonist benzodiazepines, inhibitory neuroactive steroids seem to have a decreased potential for tolerance and abuse, but only to locomotor effects. Also, when compared to benzodiazepines, inhibitory neurosteroids exhibit less effect on locomotor coordination in combination with ethanol. These data suggest that inhibitory neurosteroids might have a better safety profile than classical sedative hypnotic drugs, like benzodiazepines or barbiturates. Inhibitory neuroactive

steroids also induce anti-aggressive behavior. The inhibitory neuroactive steroids seem to decrease depressive mood. The antidepressant effect of systemically applied allopregnenolone and other α reduced neurosteroids have been repeatedly reported in different animal models of depression – forced swimming test, olfactory bulbectomy, and social isolation test [93]. Moreover, results of recent preclinical and clinical studies, connected pregnane neurosteroids (allopregnanolone and alloTHDOC) and pregnenolone with the pathophysiology of bipolar disease [94] and schizophrenia [95]. Inhibitory neuroactive steroids also modulate the acute and chronic stress reaction [96]. Brain levels of inhibitory neuroactive steroids rise during acute stress, which is followed by a decrease in HPA activity, suggesting a protective role of inhibitory neurosteroids in acute stress. On the contrary, brain levels of inhibitory neuroactive steroids decrease during chronic stress, which is followed by increased HPA activity. Excitatory neuroactive steroids induce dose dependent spectra of activity, which includes analeptic, anxiogenic (high dose), and proconvulsive actions [92]. These neurosteroids also have anamnestic (cognitive enhancing) effects [97]. The profile of behavioral activities induced by excitatory neurosteroids could be compared with that of partial inverse agonists to the benzodiazepine modulatory site. Excitatory neuroactive steroids (PS, DHEAS and DHEA) decrease depressive mood and clearly induce an antidepressant action in animals and humans [92]. They also induce aggressive behavior, impulsivity, and stimulate “acute stress like” behaviors (anxiety and excitation). In some countries, DHEA supplementation in elderly people has been advertised as an anti-aging medication [98]. Neurosteroids play an important role in alcohol tolerance and withdrawal [92, 11, 99]. In addition to the behavioral effects mentioned above, neurosteroids are also able to induce analgesic [100], anti-inflammatory [101], and neuroprotective [102, 103] effects.

5. Therapeutic potentials of synthetic GABAA modulatory steroids (GAMS) and GABAA modulatory steroid antagonists (GAMSA) Synthetic GAMS may represent a rational alternative approach to therapy with endogenous neurosteroids. One problem with natural neurosteroids is low oral


65

Tvrdeić et al.

bioavailability, due to rapid hepatic oxidation and inactivation. Another problem is their potential for unwanted, genomic effects. Namely, natural neurosteroids could be converted to precursor steroids (progesterone, testosterone), which have a high affinity for classical hormonal receptors and low affinity for neurosteroid binding sites at GABAA receptors. The neurosteroid ganaxolon is an example of a synthetic steroid, which seems to be able to overcome the mentioned problems above. Ganaxolon is a 3β -methylated synthetic analog of the endogenous neuroactive steroid allopregnanolone. It is well known that 3β- substitution enhances the bioavailability of pregnane steroids, without altering their primary pharmacodynamic properties. Ganaxolon is a positive allosteric modulator at GABAA receptors with high potency for receptors and high efficacy against chemically induced convulsions (bicuculline, pentylentetrazole, aminophylline), maximal electroshock and amigdala kindling seizures in animals [104]. A pilot study evaluating ganaxolon efficacy and safety in pediatric patients with refractory epilepsy published by Pieribone and coworkers, suggested a significant antiepileptic efficacy and good tolerability of ganaxalone in humans [105]. Basic search of the web site ClinicalTrials. gov for “Ganaxolon” made on the 11th of January 2016 yielded 11 heats. According to the ClinicvalTrials.gov web site, ganaxolon is under clinical investigation for the treatment of different forms of epilepsy: infantile spasms in children (https://ClinicalTrials.gov/show/ identifier: NCT00441896), catamenial epilepsy (https:// ClinicalTrials.gov/show/ identifier: NCT00465517), resistant partial (https://ClinicalTrials.gov/show/ identifier: NCT01963208) and complex partial (https:// ClinicalTrials.gov/show/ identifier: NCT01002820) epilepsy in adults. Also, it is under clinical investigation for the treatment of PTSD (https://ClinicalTrials.gov/show/ identifier: NCT01339689), fragile X syndrome (https:// ClinicalTrials.gov/show/ identifier: NCT01725152) and smoking cessation (https://ClinicalTrials.gov/show/ identifier: NCT01857531). GR3027 and UC1010 are investigational GABAA receptor modulating steroid antagonists (GAMSA). Both are 3β-hydroxy steroids and, as such, they are able to antagonize the enhancement of GABAA receptor activation caused by 3α-hydroxy neurosteroids. Increasing signaling through the GABAA receptor is a key driver for the neurological symptoms associated with hepatic


encephalopathy (HE) as well as for CNS symptoms (depression, anxiety) associated with the premenstrual dysphoric disorder (PMDD). GR3027 and UC1010 are promising candidate drugs intended to treat the symptoms of patients with HE or PMDD, respectively [106, 107]. Also, investigational GAMSA drugs are 17PA (3α5α-17-phenylandrost-16-en-3-ol) and HBAO (20R-17β-(1-hydroxy-2,3-butadienyl)-5α-androstane3α-ol). Both, 17PA and HBAO are synthetic derivatives and antagonists of allopregnenolone [107, 108], with relative selectivity for α6β2–3δ GABAA receptors (HBAO) or for α1β2γ2 GABAA receptors (17PA). Basic search of the web site ClinicalTrials.gov for “GR3027”, or “UC1010” or “HBAO” made on the 12th of January 12 2016 yielded only 1 heat. The study is in phase I/II, and its aim is to evaluate UC1010 (https://ClinicalTrials.gov/ show/ identifier: NCT01875718) treatment in premenstrual dysphoric disorder (PMDD).

6. Therapeutic potential of TSPO activators By definition, an “ideal” anxiolytic drug is anxioselective anxiolytic. It induces rapid and selective anxiolytic effect, without significant sedative, hypnotic, amnestic, or myorelaxant effect. Also, anxioselective anxiolytics should not have significant abuse potential or the ability to induce tolerance during long-term therapy. In the last two decades of the 20th century, a substantial amount of money was invested in the pharmaceutical industry for research and development of anxioselective drugs. But, there is no result of this investment [109]. With time, the idea of anxioselective anxiolytics became the “holy grail” of anxiolytic research and therapy [110]. The pharmacological properties of nonsteroid TSPO activators emapunil (XBD173 or AC-5216) and etifoxine seem to match the above-mentioned definition of an “ideal” anxioselective drugs. Both, emapunil (N-benzylN-ethyl-2-(7-methyl-8-oxo-2-phenylpurin-9-yl)acetamide) and etifoxine (2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride) are synthetic, nonsteroidal TSPO agonists. As such, emapunil as well as etifoxine, stimulate the synthesis of neurosteroids. Emapunil exhibited high affinity for TSPO (previously known as peripheral benzodiazepine receptors) in the crude mitochondrial fraction prepared from the whole brain of rats (Ki = 0.297 nM), but only negligible affinity for central benzodiazepine receptors [111]. In work reported by Kita and Furukawa, the anxiolytic-like

Tvrdeić et al.

effects of emapunil were inhibited by trilostane (Figure 1., 3β-HSD inhibitor), finasteride (Fig. 1, 5-α reductase inhibitor), and picrotoxin (blocker of GABAA receptor associated chloride channel), while those of diazepam (full agonist at benzodiazepine binding site) were inhibited by picrotoxin only [112]. These results clearly demonstrate that the anxiolytic-like effects of emapunil are due to newly synthesized inhibitory neurosteroids that enhance GABAA receptor function. Emapunil produces anti-anxiety and antidepressant-like effects that are mediated by TSPO and increased synthesis of inhibitory neurosteroids [111]. However, it does not cause myorelaxant, sedative/hypnotic effect, or addiction with repeated dosing [113], the side effects usually associated with conventional benzodiazepines. Like emapunil, etifoxine activates TSPO and rapidly increases the production of neurosteroids [114], especially allopregnanolone [115]. The modulatory action of etifoxine on GABAA receptor is mediated by the β subunit [116]. In addition to behavioral actions, TSPO activators have analgesic effects [117, 118], as well as neuroprotective, and anti-inflammatory actions in experimental models for neurodegenerative diseases [119-121] and neurotrauma [122]. Basic search of web site ClinicalTrials.gov for “XBD173”, or “emapunil” or “AC5216” made on the 12th of January 2016 yielded 4 heats. There is only one ongoing clinical trial (phase II) with the nonsteroid TSPO agonist emapunil for the treatment of generalized anxiety disorder (GAD). Three clinical trials with emapunil are aimed at investigating emapunil as a ligand for positron emition tomography (PET). In one of these studies, emapunil is intended for use in brain imaging of neurodegenerative diseases. Search of the same web site with the term “Etifoxine” gave another two hits. Both are Phase III studies. One study recruits volunteers aged between 65 and 75 years old (https://ClinicalTrials.gov/show/ identifier:NCT02143895) and another recruits healthy volunteers (https://ClinicalTrials.gov/show/ identifier:NCT02147548). The studies are evaluating the effects of 100 mg of Etifoxine and 2 mg of Lorazepam on vigilance and cognitive function in the elderly.

7. Conclusions Neurosteroids are important endogenous modulators of neurons and glial cell activity. They are implicated in the pathophysiology of clinically important diseases, like

anxiety, unipolar depression, postmenstrual dysphoria syndrome, bipolar depression, schizophrenia, hepatic encephalopathy, epilepsy, painful states, and brain injury. In search for ethiopharamcological approaches in treating these diseases, neurosteroids should be seriously taken into account. Neurosteroid-based drugs produce a full spectrum of pharmacological actions that overlap with those of other positive allosteric modulators of the GABA-A receptor but, at the same time, exhibit important quantitative and qualitative differences. Preclinical evaluations of GAMS, GAMSA, or TSPO activators have predicted the efficacy of these drugs in the treatment of several important CNS disorders. It seems that with these compounds, new and exciting therapeutic avenues might be opened.

Author contributions Both authors contributed in manuscript drafting, writing and revising it critically for its scientific content. Both authors have approved the final version of article.

References 1. Baulieu E-E, Robel P. Neurosteroids: A new brain function? J Steroid Biochem Mol Biol 1990;3:395–403. http://dx.doi.org/10.1016/0960-0760(90)90490-C 2. Agís-Balboa RC, Pinna G, Zhubi A, Maloku E, Veldic M, Costa E, et al. Characterization of brain neurons that express enzymes mediating neurosteroid biosynthesis. Proc Natl Acad Sci U S A 2006;103:14602–7. http://dx.doi.org/10.1073/pnas.0606544103 3. Cascio C, Brown RC, Liu Y, Han Z, Hales DB, Papadopoulos V. Pathways of dehydroepiandrosterone formation in rat brain glia. J Steroid Biochem Mol Biol 2000;75(2-3):177–86. http://dx.doi.org/10.1016/S0960-0760(00)00163-1 4. Papadopoulos V, Amri H, Boujrad N, Cascio C, Culty M, Garnier M, et al. Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis. Steroids 1997;62:21–8. http://dx.doi.org/10.1016/S0039-128X(96)00154-7 5. Vallée M, Mayo W, Le Moal M. Role of pregnenolone, dehydroepiandrosterone and their sulfate esters on learning and memory in cognitive aging. Brain Res Rev 2001;37:301–12. http://dx.doi.org/10.1016/S0165-0173(01)00135-7 6. Mellon SH, Griffin LD. Neurosteroids: Biochemistry and clinical significance. Trends Endocrinol Metab 2002;13:35–43. http://dx.doi.org/10.1016/S1043-2760(01)00503-3 7. Mensah-Nyagan G, Do-Rego JL, Beaujean D, Luu-The V, Pelletier G, Vaudry H. Neurosteroids: expression of steroidogenic enzymes and regulation of steroid biosynthesis in the central nervous system. Pharmacol Rev 1999;51:63–81.


67

Tvrdeić et al.

8. Rego JL Do, Seong JY, Burel D, Leprince J, Vaudry D, Luu-The V, et al. Regulation of neurosteroid biosynthesis by neurotransmitters and neuropeptides. Front Endocrinol (Lausanne) 2012;3:1–15. http://dx.doi.org/10.3389/fendo.2012.00004

22. Morrow AL. Recent developments in the significance and therapeutic relevance of neuroactive steroids - Introduction to the special issue. Pharmacol Ther 2007;116:1–6. http://dx.doi.org/10.1016/j.pharmthera.2007.04.003

9. Vaňková M, Hill M, Velíková M, Včelák J, Vacínová G. Reduced Sulfotransferase SULT2A1 Activity in Patients With Alzheimer’ s Disease. Physiol Res 2015;64.

23. Reddy DS. Is there a physiological role for the neurosteroid THDOC in stress-sensitive conditions? Trends Pharmacol Sci 2003;24:103–6. http://dx.doi.org/10.1016/S0165-6147(03)00023-3

10. Harteneck C. Pregnenolone sulfate: From steroid metabolite to TRP channel ligand. Molecules 2013;18:12012–28. http://dx.doi.org/10.3390/molecules181012012

24. Reddy DS. Physiological role of adrenal deoxycorticosterone-derived neuroactive steroids in stress-sensitive conditions. Neuroscience 2006;138:911–20. http://dx.doi.org/10.1016/j.neuroscience.2005.10.016

11. Hulin MW, Amato RJ, Porter JR, Filipeanu CM, Winsauer PJ. Neurosteroid Binding Sites on the GABAA Receptor Complex as Novel Targets for Therapeutics to Reduce Alcohol Abuse and Dependence. Adv Pharmacol Sci 2011;2011:1–12. http://dx.doi.org/10.1155/2011/926361

25. Finn DA, Beadles-Bohling AS, Beckley EH, Ford MM, Gililland KR, Gorin-Meyer RE, et al. A New Look at the 5 á -Reductase Inhibitor Finasteride. CNS Drug Rev 2006;12:53–76. http://dx.doi.org/10.1111/j.1527-3458.2006.00053.x

12. Maninger N, Wolkowitz OM, Reus WI ES and MS. Neurobiological and Neuropsychiatric Effects of Dehydroepiandrosterone (DHEA) and DHEA Sulfate (DHEAS). Front Neuroendocr 2009;30:65–91. http://dx.doi.org/10.1016/j.yfrne.2008.11.002

26. Belelli D, Herd MB. The contraceptive agent Provera enhances GABA(A) receptor-mediated inhibitory neurotransmission in the rat hippocampus: evidence for endogenous neurosteroids? J Neurosci 2003;23:10013–20.

13. Jin Y, Penning TM. Steroid 5alpha-reductases and 3alphahydroxysteroid dehydrogenases: key enzymes in androgen metabolism. Best Pract Res Clin Endocrinol Metab 2001;15:79–94. http://dx.doi.org/10.1053/beem.2001.0120

27. Traish AM, Mulgaonkar A, Giordano N. The dark side of 5α-reductase inhibitors’ therapy: Sexual dysfunction, high gleason grade prostate cancer and depression. Korean J Urol 2014;55:367–79. http://dx.doi.org/10.4111/kju.2014.55.6.367

14. Kaminski RM, Marini H, Kim W-J, Rogawski M. Anticonvulsant activity of androsterone and etiocholanolone. Epilepsia 2005;46:819–27. http://dx.doi.org/10.1111/j.1528-1167.2005.00705.x

28. Römer B, Gass P. Finasteride-induced depression: new insights into possible pathomechanisms. J Cosmet Dermatol 2010 Dec;9:331–2. http://dx.doi.org/10.1111/j.1473-2165.2010.00533.x

15. Kaminski RM, Marini H, Ortinski PI, Vicini S, Rogawski MA. The pheromone androstenol (5 alpha-androst-16-en-3 alpha-ol) is a neurosteroid positive modulator of GABAA receptors. J Pharmacol Exp Ther 2006;317:694–703. http://dx.doi.org/10.1124/jpet.105.098319

29. Westhoff C, Truman C, Kalmuss D, Cushman L, Davidson A, Rulin M, et al. Depressive symptoms and Depo-Provera®. Contraception 1998;57:237–40. http://dx.doi.org/10.1016/S0010-7824(98)00024-9

16. Dufort I, Soucy P, Lacoste L, Luu-The V. Comparative biosynthetic pathway of androstenol and androgens. J Steroid Biochem Mol Biol 2001;77:223–7. http://dx.doi.org/10.1016/S0960-0760(01)00057-7 17. Prelog V and Ruzicka L. Untersuchungen über Organ Extracte. 5 Mitteilung. Uber zwei moschusartig riechende Steroiden aus Schweinetestes-extracten. Helv Chim Acta 1944;27:61–6. http://dx.doi.org/10.1002/hlca.19440270108 18. Selye H. Anesthetic Effect of Steroid Hormones. Exp Biol Med 1941;46:116–21. http://dx.doi.org/10.3181/00379727-46-11907 19. Wang M, Lundgren P, Stromberg J. Allopregnanolone-stimulated GABA-mediated chloride ion flux is inhibited by 3 b -hydroxy-5 a -pregnan-20-one ( isoallopregnanolone). Brain res 2003;982:45–53. http://dx.doi.org/10.1016/S0006-8993(03)02939-1 20. Backstrom T, Wahlstrom G, Wahlstrom K, Zhu D, Wang MD. Isoallopregnanolone; an antagonist to the anaesthetic effect of allopregnanolone in male rats. Eur J Pharmacol 2005;512:15–21. http://dx.doi.org/10.1016/j.ejphar.2005.01.049 21. Guidotti A, Dong E, Matsumoto K, Pinna G, Rasmusson AM, Costa E. The socially-isolated mouse : a model to study the putative role of allopregnanolone and 5a-dihydroprogesterone in psychiatric disorders. Brain Res Brain Res Rev 2001;37:110–5. http://dx.doi.org/10.1016/S0165-0173(01)00129-1


30. Civic D, Scholes D, Ichikawa L, LaCroix AZ, Yoshida CK, Ott SM, et al. Depressive symptoms in users and non-users of depot medroxyprogesterone acetate. Contraception 2000;61:385–90. http://dx.doi.org/10.1016/S0010-7824(00)00122-0 31. Gross B, Mindea S, Pick AJ, Chandler JP, Batjer HH. Medical management of Cushing disease. Neurosurg Focus 2007;23:1–6. http://dx.doi.org/10.3171/foc.2007.23.3.12 32. Loose DS, Kan PB, Hirst M, Marcus R, Feldman D. Ketoconazole blocks adrenal steroidogenesis by inhibiting cytochrome P450-dependent enzymes. J Clin Invest 1983;71:1495–9. http://dx.doi.org/10.1172/JCI110903 33. Yin L, Hu Q. CYP17 inhibitors—abiraterone, C17,20-lyase inhibitors and multi-targeting agents. Nat Rev Urol 2014;11:32–42. http://dx.doi.org/10.1038/nrurol.2013.274 34. Penland SN, Morrow AL. 3alpha,5beta-Reduced cortisol exhibits antagonist properties on cerebral cortical GABA(A) receptors. Eur J Pharmacol 2004;506:129–32. http://dx.doi.org/10.1016/j.ejphar.2004.11.007 35. de Gier J, Wolthers CHJ, Galac S, Okkens AC, Kooistra HS. Effects of the 3β-hydroxysteroid dehydrogenase inhibitor trilostane on luteal progesterone production in the dog. Theriogenology 2011;75:1271–9. http://dx.doi.org/10.1016/j.theriogenology.2010.11.041 36. Santen RJ, Brodie H, Simpson ER, Siiteri PK, Brodie A. History of aromatase: saga of an important biological mediator and therapeutic target. Endocr Rev 2009;30:343–75. http://dx.doi.org/10.1210/er.2008-0016

Tvrdeić et al.

37. Reddy DS. Testosterone modulation of seizure susceptibility is mediated by neurosteroids 3alpha-androstanediol and 17beta-estradiol. Neuroscience 2004;129:195–207. http://dx.doi.org/10.1016/j.neuroscience.2004.08.002

51. Atack JR. The benzodiazepine binding site of GABAA receptors as a target for the development of novel anxiolytics. Expert Opin Investig Drugs 2005;14:601–18. http://dx.doi.org/10.1517/13543784.14.5.601

38. Pinna G, Costa E GA. SSRIs act as selective brain steroidogenic stimulants (SBSSs) at low doses that are inactive on 5-HT reuptake. Curr Opin Pharmacol 2009;9:24–30. http://dx.doi.org/10.1016/j.coph.2008.12.006

52. Olsen RW, Chang C-SS, Li G, Hanchar HJ, Wallner M. Fishing for allosteric sites on GABAA receptors. Biochem Pharmacol 2004;68:1675–84. http://dx.doi.org/10.1016/j.bcp.2004.07.026

39. Pinna G, Costa E, Guidotti A. Fluoxetine and norfluoxetine stereospecifically and selectively increase brain neurosteroid content at doses that are inactive on 5-HT reuptake. Psychopharmacology (Berl) 2006 Jun;186:362–72. http://dx.doi.org/10.1007/s00213-005-0213-2 40. Pinna G. In a mouse model relevant for post-traumatic stress disorder, selective brain steroidogenic stimulants (SBSS) improve behavioral deficits by normalizing allopregnanolone biosynthesis. Behav Pharmacol 2010 Sep;21:438–50. http://dx.doi.org/10.1097/FBP.0b013e32833d8ba0 41. Rupprecht R, Papadopoulos V, Rammes G, Baghai TC, Fan J, Akula N, et al. Translocator protein (18 kDa) (TSPO) as a therapeutic target for neurological and psychiatric disorders. Nat Rev Drug Discov 2010;9:971–88. http://dx.doi.org/10.1038/nrd3295 42. Miller PS, Aricescu AR. Crystal structure of a human GABAA receptor. Nature 2014;512:270–5. http://dx.doi.org/10.1038/nature13293 43. Karlin A, Akabas MH. Toward a structural basis for the function of nicotinic acetylcholine receptors and their cousins. Neuron 1995 Dec;15:1231–44. http://dx.doi.org/10.1016/0896-6273(95)90004-7 44. Martini C, Rigacci T, Lucacchini A. [ 3 H]Muscimol Binding Site on Purified Benzodiazepine Receptor. J Neurochem 1983 Oct;41:1183–5. http://dx.doi.org/10.1111/j.1471-4159.1983.tb09070.x

53. thompson SA, Whiting PJ, Wafford KA. Barbiturate interactions at the human GABAA receptor: dependence on receptor subunit combination. Br J Pharmacol 1996 Feb;117:521–7. http://dx.doi.org/10.1111/j.1476-5381.1996.tb15221.x 54. Fisher MT, Fisher JL. Activation of alpha6-containing GABAA receptors by pentobarbital occurs through a different mechanism than activation by GABA. Neurosci Lett 2010 Mar 8;471:195–9. http://dx.doi.org/10.1016/j.neulet.2010.01.041 56. Kalueff V. Mapping convulsants’ binding to the GABA-A receptor chloride ionophore: a proposed model for channel binding sites. Neurochem Int 2007;50:61–8. http://dx.doi.org/10.1016/j.neuint.2006.07.004 57. Leeb-Lundberg F, Napias C, Olsen RW. Dihydropicrotoxinin binding sites in mammalian brain: Interaction with convulsant and depressant benzodiazepines. Brain Res 1981 Jul;216:399–408. http://dx.doi.org/10.1016/0006-8993(81)90141-4 58. Hosie AM, Wilkins ME, Smart TG. Neurosteroid binding sites on GABAA receptors. Pharmacol Ther 2007;116:7–19. http://dx.doi.org/10.1016/j.pharmthera.2007.03.011 59. Ahboucha S, Desjardins P, Chatauret N, Pomier-Layrargues G, Butterworth RF. Normal coupling of brain benzodiazepine and neurosteroid modulatory sites on the GABA-A receptor complex in human hepatic encephalopathy. Neurochem Int 2003;43:551–6. http://dx.doi.org/10.1016/S0197-0186(03)00065-2

45. Baumann SW, Baur R, Sigel E. Individual properties of the two functional agonist sites in GABA(A) receptors. J Neurosci 2003;23:11158–66.

60. Svob Štrac D, Jazvinšćak Jembrek M, Erhardt J, Mirković Kos K, Peričić D. Modulation of recombinant GABA(A) receptors by neurosteroid dehydroepiandrosterone sulfate. Pharmacology 2012 Jan;89:163–71. http://dx.doi.org/10.1159/000336058

46. Deng L, Ransom RW, Olsen RW. [3H]Muscimol photolabels the γ-aminobutyric acid receptor binding site on a peptide subunit distinct from that labeled with benzodiazepines. Biochem Biophys Res Commun 1986 Aug;138:1308–14. http://dx.doi.org/10.1016/S0006-291X(86)80425-9

61. Sousa A, Ticku MK. Interactions of the neurosteroid dehydroepiandrosterone sulfate with the GABA(A) receptor complex reveals that it may act via the picrotoxin site. J Pharmacol Exp Ther 1997;282:827–33.

47. Bujas M, Tvrdeić A, Peričić D. Regional differences in NaCl-induced increase of the potency of bicuculline to displace [3H]muscimol binding. Neurochem Int 1997;30:199–202. http://dx.doi.org/10.1016/S0197-0186(96)00054-X 48. Sigel E, Buhr A. The benzodiazepine binding site of GABA(A) receptors. Trends Pharmacol Sci 1997;18:425–9. http://dx.doi.org/10.1016/S0165-6147(97)90675-1 49. Berezhnoy D, Nyfeler Y, Gonthier A, Goeldner M, Sigel E. On the Benzodiazepine Binding Pocket in GABA A Receptors. Biochemistry 2004;279:3160–8. 50. Buhr a, Sigel E. A point mutation in the gamma2 subunit of gamma-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity. Proc Natl Acad Sci U S A 1997;94:8824–9. http://dx.doi.org/10.1073/pnas.94.16.8824

62. Bowery N, Enna SJ, Olsen RW. Six decades of GABA. Biochem Pharmacol 2004;68:1477–8. http://dx.doi.org/10.1016/j.bcp.2004.07.033 63. Krnjević K. How does a little acronym become a big transmitter? Biochem Pharmacol 2004;68:1549–55. http://dx.doi.org/10.1016/j.bcp.2004.06.038 64. Bowery NG, Smart TG. GABA and glycine as neurotransmitters: a brief history. Br J Pharmacol 2006;147 Suppl :S109–19. http://dx.doi.org/10.1038/sj.bjp.0706443 65. Olsen R, Peters JA, Hales TG, Sieghart W, Rudolph U, Lambert JJ, et al. GABAA receptors [Internet]. IUPHAR/BPS Guide to PHARMACOLOGY. 2015. Available from: http://www. guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=7 66. Sieghart W. Structure and Pharmacology Receptor of Subtypes AcidA. Pharmacol Rev1995;47:182–224.


69

Tvrdeić et al.

67. Li G-D, Chang C-SS, Olsen RW. Anesthetic sites on GABAA receptors. Int Congr Ser 2005;1283:61–6. http://dx.doi.org/10.1016/j.ics.2005.06.054 68. Forman SA, Miller KW. Anesthetic sites and allosteric mechanisms of action on Cys-loop ligand-gated ion channels. Can J Anaesth 2011;58:191–205. http://dx.doi.org/10.1007/s12630-010-9419-9 69. Olsen RW, Hanchar HJ, Meera P, Wallner M. GABAA Receptor Subtypes: the “One Glass of Wine” Receptors. Alcohol 2007;41:201–9. http://dx.doi.org/10.1016/j.alcohol.2007.04.006 70. Olsen RW, Li G-D, Wallner M, Trudell JR, Bertaccini EJ, Lindahl E, et al. Structural models of ligand-gated ion channels: sites of action for anesthetics and ethanol. Alcohol Clin Exp Res 2014;38:595–603. http://dx.doi.org/10.1111/acer.12283 71. Korpi ER, Lüddens H. Furosemide interactions with brain GABAA receptors. Br J Pharmacol 1997 Feb;120:741–8. http://dx.doi.org/10.1038/sj.bjp.0700922 72. Minier F, Sigel E. Positioning of the alpha-subunit isoforms confers a functional signature to gamma-aminobutyric acid type A receptors. Proc Natl Acad Sci U S A 2004;101:7769–74. http://dx.doi.org/10.1073/pnas.0400220101 73. Horenstein J, Akabas MH. Location of a High Affinity Zn2+ Binding Site in the Channel of alpha 1beta 1 gamma -Aminobutyric AcidA Receptors. Mol Pharmacol 1998;53:870–7. 74. Yakushiji T, Shirasaki T, Akaike N. Non-competitive inhibition of GABAA responses by a new class of quinolones and non-steroidal anti-inflammatories in dissociated frog sensory neurones. Br J Pharmacol 1992;105:13–8. http://dx.doi.org/10.1111/j.1476-5381.1992.tb14203.x 75. Coyne L, Su J, Patten D, Hakkiwell RF. Characterization of the Interaction between Fenamates and Hippocampal Neuron GABAA Receptors. Neurochem Int 2007;51:440–6. http://dx.doi.org/10.1016/j.neuint.2007.04.017 76. Tvrdeić A, Peričić D. Dihydrogenated ergot compounds bind with high affinity to GABAA receptor associated Cl- ionophore. Eur J Pharmacol 1991;202:109–11. http://dx.doi.org/10.1016/0014-2999(91)90262-O 77. Tvrdeić A, Peričić D. Dihydroergotoxine modulation of the GABAA receptor-associated Cl- ionophore in mouse brain. Eur J Pharmacol 1992;221:139–43. http://dx.doi.org/10.1016/0014-2999(92)90783-Z 78. Tvrdeić A, Peričić D. Effect of ergot alkaloids on 3H-flunitrazepam binding to mouse brain GABAA receptors. Coll Antropol 2003;27 Suppl 1:175–82. 79. The 2015 Nobel Prize in Physiology or Medicine - Press Release [Internet]. Nobelprize.org. Nobel Media AB 2014. [cited 2016 Jan 9]. Available from: http://www.nobelprize.org/nobel_prizes/medicine/ laureates/2015/press.html 80. Pong SS, DeHaven R, Wang CC. A comparative study of avermectin B1a and other modulators of the gamma-aminobutyric acid receptor chloride ion channel complex. J Neurosci 1982;2:966–71. 81. Estrada-Mondragon A, Lynch JW. Functional characterization of ivermectin binding sites in α1β2γ2L GABA(A) receptors. Front Mol Neurosci 2015;8:55. http://dx.doi.org/10.3389/fnmol.2015.00055


82. Barnard E, Skolnick P, Olsen R, Mohler H, Sieghart W, Biggio G, et al. International Union of Pharmacology. XV. Subtypes of gammaaminobutyric acidA receptors: classification on the basis of subunit structure and receptor function. Pharmacol Rev 1998;50:291–313. 83. Ollsen RW, Sieghart W. International Union of Pharmacology. XV. Subtypes of gamma-aminobutyric acidA receptors: classification on the basis of subunit structure and receptor function. Pharmacol Rev 2008;60:243–60. http://dx.doi.org/10.1124/pr.108.00505 84. Majewska MD, Demirgoren S, Spivak CE, London ED. The neurosteroid dehydroepiandrosterone sulfate is an allosteric antagonist of the GABAA receptor. Brain Res 1990;526:143–6. http://dx.doi.org/10.1016/0006-8993(90)90261-9 85. Majewska MD, Harrison NL, Schwartz RD, Barker JL PS. Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor. Science 1986;232:1004–7. http://dx.doi.org/10.1126/science.2422758 86. Harrison NL, Majewska MD, Harrington JW BJ. Structure-activity relationships for steroid interaction with the gamma-aminobutyric acid A receptor complex. J Pharmacol Exp Ther 1987;241:346–53. 87. Hosie AM, Wilkins ME, da Silva HMA, Smart TG. Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites. Nature 2006;444:486–9. http://dx.doi.org/10.1038/nature05324 88. Hosie AM, Clarke L, da Silva H, Smart TG. Conserved site for neurosteroid modulation of GABAA receptors. Neuropharmacology 2009;56:149–54. http://dx.doi.org/10.1016/j.neuropharm.2008.07.050 89. Mitchell EA, Herd MB, Gunn BG, Lambert JJ, Belelli D. Neurosteroid modulation of GABAA receptors: Molecular determinants and significance in health and disease. Neurochem Int 2008;52:588–95. http://dx.doi.org/10.1016/j.neuint.2007.10.007 90. Smith SS, Shen H, Gong QH, Zhou X. Neurosteroid regulation of GABAA receptors: Focus on the α4 and δ subunits. Pharmacol Ther 2007;116:58–76. http://dx.doi.org/10.1016/j.pharmthera.2007.03.008 91. Garcia-Borreguero D. Article reviewed : Attenuated sensitivity to neuractive steroids in gamma-aminobutyrate type A receptor delta subunit knockout mice. Sleep Med 2000;1:159–60. http://dx.doi.org/10.1016/S1389-9457(00)00017-4 92. Reddy D. Neurosteroids: Endogenous Role in the Human brain and Therapeutic Potentials. Prog Brain Res 2010;186:113–37. http://dx.doi.org/10.1016/B978-0-444-53630-3.00008-7 93. Uzunova V, Sampson L, Uzunov DP. Relevance of endogenous 3α-reduced neurosteroids to depression and antidepressant action. Psychopharmacology (Berl) 2006;186:351–61. http://dx.doi.org/10.1007/s00213-005-0201-6 94. Carta MG, Bhat KM, Preti A. GABAergic neuroactive steroids: a new frontier in bipolar disorders? Behav Brain Funct 2012;8:61. http://dx.doi.org/10.1186/1744-9081-8-61 95. Marx CE, Bradford DW, Hamer RM, Naylor JC, Allen TB, Lieberman JA, et al. Pregnenolone as a novel therapeutic candidate in schizophrenia: emerging preclinical and clinical evidence. Neuroscience 2011;191:78–90. http://dx.doi.org/10.1016/j.neuroscience.2011.06.076

Tvrdeić et al.

96. Gunn BG, Cunningham L, Mitchell SG, Swinny JD, Lambert JJ, Belelli D. GABAA receptor-acting neurosteroids: A role in the development and regulation of the stress response. Front Neuroendocrinol 2015;36:28–48. http://dx.doi.org/10.1016/j.yfrne.2014.06.001 97. Wolf OT, Kirschbaum C. Actions of dehydroepiandrosterone and its sulfate in the central nervous system: effects on cognition and emotion in animals and humans. Brain Res Brain Res Rev 1999;30:264–88. http://dx.doi.org/10.1016/S0165-0173(99)00021-1

109. Kinch MS. An analysis of FDA-approved drugs for pain and anesthesia. Drug Discov Today 2015;20:3–6. http://dx.doi.org/10.1016/j.drudis.2014.09.002 110. Skolnick P. Anxioselective anxiolytics: On a quest of holly grail. Trends Pharmacol Sci 2012;33:611–20. http://dx.doi.org/10.1016/j.tips.2012.08.003

98. Allolio B, Arlt W. DHEA treatment : myth or reality? Trends Endocrinol Metab 2002;13:288–94. http://dx.doi.org/10.1016/S1043-2760(02)00617-3

111. Kita A, Kohayakawa H, Kinoshita T, Ochi Y, Nakamichi K, Kurumiya S, et al. Antianxiety and antidepressant-like effects of AC-5216, a novel mitochondrial benzodiazepine receptor ligand. Br J Pharmacol 2004 Aug;142:1059–72. http://dx.doi.org/10.1038/sj.bjp.0705681

99. Biggio G, Concas A, Follesa P, Sanna E, Serra M. Stress, ethanol, and neuroactive steroids. Pharmacol Ther 2007;116:140–71. http://dx.doi.org/10.1016/j.pharmthera.2007.04.005

112. Kita A, Furukawa K. Involvement of neurosteroids in the anxiolyticlike effects of AC-5216 in mice. Pharmacol Biochem Behav 2008;89:171–8. http://dx.doi.org/10.1016/j.pbb.2007.12.006

100. Poisbeau P, Keller AF, Aouad M, Kamoun N, Groyer G, Schumacher M. Analgesic strategies aimed at stimulating the endogenous production of allopregnanolone. Front Cell Neurosci 2014:1–8. http://dx.doi.org/10.3389/fncel.2014.00174

113. Kita A, Kinoshita T, Kohayakawa H, Furukawa K, Akaike A. Lack of tolerance to anxiolysis and withdrawal symptoms in mice repeatedly treated with AC-5216, a selective TSPO ligand. Prog Neuropsychopharmacol Biol Psychiatry 2009;33:1040–5. http://dx.doi.org/10.1016/j.pnpbp.2009.05.018

101. Gutiérrez G, Mendoza C, Zapata E, Montiel A, Reyes E, Monta-o LF, et al. Dehydroepiandrosterone inhibits the TNF-alpha-induced inflammatory response in human umbilical vein endothelial cells. Atherosclerosis 2007;190:90–9. http://dx.doi.org/10.1016/j.atherosclerosis.2006.02.031 102. Lazaridis I, Charalampopoulos I, Alexaki V-I, Avlonitis N, Pediaditakis I, Efstathopoulos P, et al. Neurosteroid Dehydroepiandrosterone Interacts with Nerve Growth Factor (NGF) Receptors, Preventing Neuronal Apoptosis. PLoS Biol 2011;9:e1001051. http://dx.doi.org/10.1371/journal.pbio.1001051

114. Verleye M, Akwa Y, Liere P, Ladurelle N, Pianos A, Eychenne B, et al. The anxiolytic etifoxine activates the peripheral benzodiazepine receptor and increases the neurosteroid levels in rat brain. Pharmacol Biochem Behav 2005;82:712–20. http://dx.doi.org/10.1016/j.pbb.2005.11.013 115. Ugale RR, Sharma AN, Kokare DM, Hirani K, Subhedar NK, Chopde CT. Neurosteroid allopregnanolone mediates anxiolytic effect of etifoxine in rats. Brain Res 2007;1184:193–201. http://dx.doi.org/10.1016/j.brainres.2007.09.041

103. Luchetti S, Huitinga I, Swaab DF. Neurosteroid and GABA-A receptor alterations in Alzheimer’s disease, Parkinson’s disease and multiple sclerosis. Neuroscience 2011;191:6–21. http://dx.doi.org/10.1016/j.neuroscience.2011.04.010

116. Hamon A, Morel A, Hue B, Verleye M, Gillardin JM. The modulatory effects of the anxiolytic etifoxine on GABAA receptors are mediated by the β subunit. Neuropharmacology 2003;45:293–303. http://dx.doi.org/10.1016/S0028-3908(03)00187-4

104. Carter RB, Wood PL, Wieland S, Hawkinson JE, Belelli D, Lambert JJ, et al. Characterization of the Anticonvulsant Properties of Ganaxolone (CCD 1042; 3α-Hydroxy-3β-methyl-5α-pregnan-20-one), a Selective, High-Affinity, Steroid Modulator of the γ-Aminobutyric AcidA Receptor. J Pharmacol Exp Ther 1997;280:1284–95.

117. Choi YM, Kim KH. Etifoxine for pain patients with anxiety. Korean J Pain 2015;28:4–10. http://dx.doi.org/10.3344/kjp.2015.28.1.4

105. Pieribone VA, Tsai J, Soufflet C, Rey E, Shaw K, Giller E, et al. Clinical evaluation of ganaxolone in pediatric and adolescent patients with refractory epilepsy. Epilepsia 2007;48:1870–4. http://dx.doi.org/10.1111/j.1528-1167.2007.01182.x 106. Johansson M, Agusti A, Llansola M, Montoliu C, Strömberg J, Malinina E, et al. GR3027 antagonizes GABA A receptor-potentiating neurosteroids and restores spatial learning and motor coordination in rats with chronic hyperammonemia and hepatic encephalopathy. Am J Physiol - Gastrointest Liver Physiol 2015;309:G400–9. http://dx.doi.org/10.1152/ajpgi.00073.2015 107. Johansson M, Strömberg J, Ragagnin G, Doverskog M, Bäckström T. GABAA receptor modulating steroid antagonists (GAMSA) are functional in vivo. pii: S0960-0760(15)30121-7. doi: 10.1016/j.jsbmb.2015.10.019. http://dx.doi.org/10.1016/j.jsbmb.2015.10.019 108. Maksay G, Fodor L, Bíró T, Avlonitis N, Calogeropoulou T. A 17beta-derivative of allopregnanolone is a neurosteroid antagonist at a cerebellar subpopulation of GABA A receptors with nanomolar affinity. Br J Pharmacol 2007;151:1078–86. http://dx.doi.org/10.1038/sj.bjp.0707316

118. Wang D, Tian Z, Guo Y, Guo H, Kang W, Li S, et al. Anxiolytic-like effects of translocator protein (TSPO) ligand ZBD-2 in an animal model of chronic pain. Mol Pain. 2015;11:1–10. http://dx.doi.org/10.1186/s12990-015-0013-6 119. Barron AM, Garcia-Segura LM, Caruso D, Jayaraman A, Lee J-W, Melcangi RC, et al. Ligand for translocator protein reverses pathology in a mouse model of Alzheimer’s disease. J Neurosci 2013;33:8891–7. http://dx.doi.org/10.1523/JNEUROSCI.1350-13.2013 120. Girard C, Liu S, Adams D, Lacroix C, Sinéus M, Boucher C, et al. Axonal regeneration and neuroinflammation: roles for the translocator protein 18 kDa. J Neuroendocrinol 2012;24:71–81. http://dx.doi.org/10.1111/j.1365-2826.2011.02215.x 121. Scholz R, Caramoy A, Bhuckory MB, Rashid K, Chen M, Xu H, et al. Targeting translocator protein (18 kDa) (TSPO) dampens proinflammatory microglia reactivity in the retina and protects from degeneration. J Neuroinflammation 2015;12:201. http://dx.doi.org/10.1186/s12974-015-0422-5 122. Papadopoulos V, Lecanu L. Translocator protein (18 kDa) TSPO: an emerging therapeutic target in neurotrauma. Changes 2009;29:997–1003. http://dx.doi.org/10.1016/j.expneurol.2009.04.016


71